The overall goal of this procedure is to observe the dynamic interaction between a single DNA binding protein and a DNA molecule. This is achieved by combining in a custom built microscope, two single molecule techniques, double optical tweezers and fluorescence microscopy. This setup is also equipped with bright field illumination for visualization and nanometer stabilization of the sample.
As a second step, a multi-channel flow cell integrated in a micro flow system is built to allow up to four laminar flows to efficiently recruit and assemble all the components required for the experiment. Next, the single molecule construct is assembled in order to perform the experiments. First two strip titin coated microspheres are caught with the dual optical tweezers in the first channel.
The two trapped beads are then moved to the second channel to allow the attachment of the DNA template labeled with biotin on both extremities. The presence of a single DNA molecule is checked in the third channel by A DNA force extension analysis. Lastly, the bead DNA bead construct named DNA dumbbell is moved to the protein channel where the fluorescently labeled lactose repressor protein is present.
Results are obtained that show diffusion and specific binding of single LAC eye molecules on a single stretched DNA molecule and the quantification of the kinetic parameters of LAC eye motion. The main advantages of this technique over existing single molecule methods are the possibility to control tension applied to the new molecule, the possibility to precisely track movements of the binding proteins over an nanometer stable spot, as well as the ability to operate far from the cover sleep surface. This method can help answer key questions on protein DNA interactions, especially on the mechanics of gene expression regulation, as well as the dynamics of protein diffusion along the DNA in the mechanisms of target search.
These demonstration of this method is very useful to show the practical aspects of the construction of the flow cell sample handling and assembly of the beat DNA bead construct. This video will first demonstrate how to check the pointing stability of the two trapping lasers using micro beads stuck on a cover slip surface as a reference. Details on the design and construction of the optical tweezer setup can be found in the accompanying protocol text.
Use a 24 by 60 millimeter cover slip to spread two microliters of silica beads onto the surface of a 24 by 32 millimeter cover slip. Using two pieces of double-sided tape. Attach the 24 by 32 millimeter cover slip onto a microscope slide to create a flow cell.
Fill the flow cell with 50 millimolar of phosphate buffer and seal it with silicon grease image. One silica bead in brightfield microscopy at approximately 2000 times magnification. The field of view should be approximately seven by five micrometers acquired at 7 68 by 5 76 pixels so that the bead image has a diameter of a approximately 190 pixels compensate thermal drifts with a feedback software that calculates XY position from the image centro and Z from the diffraction rings and corrects drifts.
Moving a pizo stage with nanometer accuracy or better overlap the center of the left trap with the bead center and measure the position noise and its standard deviation from the quadrant detector photo diode or QDP. Repeat the measurement for the right trap. The standard deviation of the position noise should be within a few nanometers to allow precise localization of DNA binding proteins to make the flow cell.
A diamond tip is first used to drill one millimeter diameter holes in a 76 by 76 by one millimeter microscope. Slide to create four inlets and one outlet. This will allow up to four parallel buffer flows.
Each containing one of the different components required for the experiment beads, DNA fluorescently labeled protein and imaging buffer. Clean the microscope slide with ethanol and center the port with the O-ring inserted and the adhesive ring in the slide surrounding the access holes. It is essential to wear gloves during this step to avoid fingerprints, which would disturb the gluing process.
With the help of a second slide clamp the nano ports to the slide and place it in an oven preheated at 180 degrees Celsius for one hour to allow complete attachment. In the meantime, draw the outline pattern of the flow cell on a piece of paper. The channel should be approximately three millimeters wide and match the whole positions on the microscope slide.
Place the drawing on top of two pieces of perfil and with a scalpel, make sharp and continuous cuts along the drawn outline. Remove any protuberances formed, discard the lower parfum layer, which is just used to guarantee a clean support. Next place the microscope slide containing the attached nano ports upside down in the metallic support.
Carefully align the paraform chamber outline with the holes, making sure that the param does not extrude the inlets and outlet of the chamber cover with a cleaned microscope cover slip and carefully remove excess params surrounding the chamber. Place two heat blocks previously heated at 120 degrees Celsius on top of the flow cell to apply homogenous pressure on it. Let the para film melt for approximately 25 minutes.
The next step is to prepare the pressure reservoir and buffer containers. The pressure reservoir is made of plexiglass and allows accommodation of up to six buffer containers. First, connect the shutoff valves at the exit of the buffer containers for turning the buffer flow on with the valve lever in the vertical position or off in the clockwise rotated position.
During the experiments, mount the flow cell on the microscope stage. Then connect the shutoff valves through the poly ether, ether ketone, or peak tubing, and the flangeless spinnings to the flow chamber inlets. The peak tubing has an inner diameter of approximately 150 micrometers kilometers, approximately three centimeters of fluorinated ethylene propylene tubing with an inner diameter of approximately 500 micrometers are used as a linkage between the peak tubing and the nano port assemblies of the flow cell.
This is essential to reduce the buffer flow at the chamber entrance to avoid breakage of the flow cell or nano port detachment from the glass. The use of large inner diameter tubing alone on the other hand, would require large volumes of biological samples. Connect the flow chamber outlet to an open Falcon tube at atmospheric pressure.
This tube serves as disposal for the buffer flowing out of the flow chamber. To begin this procedure, add one milliliter of buffer A to each of the buffer containers and apply 200 millibars of pressure to wash the connecting tubes and flow cell. A pressure control system is capable.
Finally, adjusting the air pressure inside the pressure reservoir. Check for the presence of air bubbles, which might disrupt the flow smoothness. A gentle flick on the outlet tube will help to remove any remaining bubbles, decrease the pressure to 20 millibars, and verify that all channels and tubes are clear from air bubbles and that the buffer flows with similar velocities in all channels.
Prepare the samples to a final volume of 300 microliters. This experiment utilizes strept coated polystyrene beads, linear DNA biotinylated on both extremities that contain specific target sequences for lactose repressor binding and fluorescently labeled lactose repressor protein. Add each sample to one of the syringe containers in the following order beads in the first channel DNA in the second, the imaging buffer in the third and the labeled protein in the fourth channel.
Turn the flow on at 20 millibars and open the shutoff valves to let the samples arrive inside the flow channel. This is easily detectable in the bead channel. While imaging the first channel under brightfield illumination, switch on the two optical tweezers and catch a polystyrene bead in each trap.
Move quickly to the second channel to avoid the other beads falling into the traps. Note that the arrival in the DNA channel is easily noticeable by the end of the bead flow. Using one AO optic deflector or a OD move, one trap bead back and forth in the proximity of the other bead to allow the attachment of the flow extended DNA in between the two optically trapped beads.
When a DNA dumbbell is formed, the static bead starts following the movement of the bead that is actively moved back and forth by the trap. Move quickly to the buffer channel and turn off the buffer flow. Perform a force extension curve analysis to verify the presence of a single DNA molecule.
This is accomplished by separating the two beads apart while measuring the DNA tension. Once a single DNA dumbbell is obtained, turn on the flow again and move the dumbbell to the protein channel. Switch to wide field fluorescence microscopy to monitor the interaction of the labeled lactose repressor protein with DNA, turn off the flow and start acquisition.
This method enables the detection of single lactose repressor or lac eye molecules interacting with a stretched DNA molecule. In this representative CH agram, a single lac eye molecule diffuses along non-specific DNA sequences, while another lac eye molecule is specifically bound to one operator located in the center of the DNA molecule. This plot shows the position of the two lac eye molecules along the direction connecting the centers of the two traps.
As a function of time, the positions of the molecules were determined by the radial symmetry center algorithm shown here is a plot of the mean square displacement or MSD versus time for a single lac eye molecule diffusing along a stretched DNA molecule. The diffusion coefficient of the protein can be readily obtained from linear regression of the data. The combination of manipulation and imaging techniques is a powerful method, allowing mechanical control on a single biological polymer such as D-N-A-R-N-A or a cytoskeletal filament, and the simultaneous localization of single protein molecules interacting with the polymer itself.
While attempting this Procedure, it's important to remember that combining single molecule manipulation and imaging techniques require careful design of the experimental setup to ensure nanometer level stability of the suspended polymer. On the other end, proper design of the biological probe is needed to guarantee special separation between the trapping G laser and the fluorescent probe. Please refer to the accompanying protocol text for details.
After watching this video, you should have a good understanding of how to assemble the DNA molecule in between the two optical rapid beats the assembly of this construct. It's better obtained with the multichannel flow cell for which we provided the detailed description. We also indicated how to optimize buffer flow and to remove the air bubbles, which otherwise could compromise your experiments.
In the combining text protocol, you can find the protocols to label the DNA with bioTE, which is necessary for the assembly with the streptavidin coated beats as well as the protocols to labeled the protein. Finally, we illustrated the step-by-step operations to quantify the binding and the diffusion of a single lactose repressive protein on a stretch DNA molecule.
